This project extends our long-standing interests in establishing a murine model to study HEV replication, pathogenesis and therapy. HIV-1 is unable to infect murine cells. Attempts to "genetically humanize" the mouse with HIV-1 receptors have yielded only limited success. HIV-1 can establish initial infection in vitro or in vivo in cells or transgenic mice expressing human CD4, CCR5 (or CXCR4), and the Tat cofactor cyclin TI, but with no sustained replication and spread. Therefore, additional human factors, and possible murine factors that inhibit HIV-1 replication, will make further humanization of the mouse for H N replication difficult. However, a highly mutable virus like HIV-1 will be able to adapt to replicate in such Genetically Humanized Mouse (GHM) cells. I propose to make HN-1 "do the work" to evolve in GHM cells both in vitro and in vivo. The putative HIV-1 variants will depend on human CD4lCCRS and Tat/cyclin TI to infect the GHM cell. The specific aims are: 1. To adapt HIV-1 to replicate in GHM cells with GHM/human cell co-cultures in vitro and in vivo. The GHM mouse has overcome three blocks for HIV-1 infection. The failure of HIV-1 to replicate efficiently in the GHM cell, either due to a lack of additional human factors or presence of negative murine factors, provides a genetic selection system for HIV-I to "evolve" in these cells. As successfully demonstrated with murine hepatitis virus (MHV), it is possible to direct host (species)-range shift by using escalating ratios of restrictive/permissive target cells. I will start a similar evolution process with CXCR4-tropic, CCR5-tropic and CXCR4/CCRS dual-tropic HIV-1 isolates in vitro in GHM and human cells. In addition, I will use GHM bone marrow (BM) cells to reconstitute SCID-hu ThyLiv mice which have been infected/depleted with HIV- 1. This allows HIV-1 adaptation from human cells to GHM cells in vivo at an increasing GHM/human cell ratio. Murine T cells developed in the HIV-1 infected ThyLiv organs will be activated in vitro to expand and recover the HIV- 1 variants. 2. To derive HIV-1 variants evolved to replicate in the GHM mouse in vivo. I will use two different approaches. First, I will inoculate HIV-1 (evolved from aim 1 or infected human cells) in the thymus of newborn GHM mice, whose immune system is actively developing (but immature) and should be permissive to viral infection. Second, I will infect the GHM BM cells (containing T, macrophage and progenitor cells) with. the evolved WN-1 (or infected human cells) and reconstitute lethally irradiated GHM mice with the HIV-infected BM cells. The HIV infection and bone marrow transplant procedure will be repeated to expand the NIV- 1 variants. A murine model for studying HIV-1 pathogenesis and host immune responses will be obviously invaluable for studying HIV-1 replication, pathogenesis and therapy. The adapted HIV-1 will infect the GHM model via human CD4 and coreceptors, and human cyclin T1. It is likely that the adapted HIV in the GHM model will lead to AIDS-like diseases because 1) human CD4 is essential for CD4 T development and function in the GI-IM mouse; 2) both CD4 T and macrophage cells are HIV targets; and 3) expression of nef in transgenic mouse models can lead to AIDS like diseases. Even if no AIDS-like pathology were developed, the GHwf model infected with the HIV-1 variants will be still useful for testing therapeutics that target HIV- 1 entry, tat- mediated gene expression and neutralizing antibody response in this model.